Photodynamic properties of toluidine blue characterized by fluorescence and absorption spectroscopy

Specmchimica Am, Vol. 46A, Printed in Great Britain

No. 8, pp. 12651267.

1990 0

TECHNICAL

Photodynamic

0584~8539/90 93.llo+0.00 1990 Pergamon Press plc

NOTE

properties of toluidine blue characterized and absorption spectrosccpy

by fluorescence

(Received 26 August 1989; in @al form 27 January 1990; accepted 31 January 1990)

Abstract-Red light photolysis studies of the production of singlet oxygen by toluidine blue dye photosensitixation were measured by the photo-oxidation rate of tryptophan. Absorption spectra of toluidine blue together with each of the four synthetic nucleosides of DNA have been obtained. After irradiation, only deoxyguanosine showed any noticeable reaction at neutral pH. Abreviations (see Fig. 1): TB, toluidine blue; ‘Or, singlet oxygen; TRP, tryptophan; dA, 2’-deoxyadenosine; dC, 2’-deoxycytidine; dT, 2’-deoxythymidine; dG, 2’-deoxyguanosine; NaN,, sodium axide.

In the present note we have studied some photodynamic properties of TB. TRP photosensitization was used to determine ‘02 production by different photosensitizers with visible light [l, 21. A significant decrease in the fluorescence intensity of TRP (5 x lo-’ M) in the presence of TB was

HO 2’-

H

HO 2’-

Deoxyadenoslne

H

Deaxyguanoslne

NH2

0

I

H

N&H

HN %-CH, 0-i

.N/tH

I

HO

HC)

H

2’-

2’ -Deuxycytldlne

Toluldlne

ti

Deoxythymldlne

Tryptcphan

blue

Fig. 1. Structural formulae of some of the species used in this work. 1265

Technical Note

1266

I 5x10-5 Concentmtion

Fig. 2. Effect

I 10-4 (Ml

of different TB concentrations on the photodegradation (5 x 10-r M) at pH 7.0 by red light irradiation.

of TRP solutions

observed when mixed solutions were irradiated for 45 min. The dependence of the photodegradation of TRP on TB concentrations (10e5 M, 5 X 10e5 M, 10m4M) is illustrated in Fig. 2. TB is formed to be optimally effective at a concentration of 10e4M. We have performed the same experiments in the presence of the azide ion N;, which is an efficient quencher of ‘02 [l, 21. Figure 3 shows the effect of increasing the concentration of NaN, on the TRP photodegradation at different irradiation times in the presence of TB (10m4M). In all cases, inibition is observed, reaching a value of 50% at an NaN3 concentration of 5 X lo-’ M with complete inhibition at 10e2 M of NaNr. On the other hand TRP buffered solutions (pH 7.0) irradiated without TB do not show any degradation. Some photosensitizers cause photodynamic oxidation of nucleic acids and their components [3,4] and it has been shown that guanine reacts much faster than any other of the DNA components under in uitro conditions [5,6]. Under prolonged illumination (6 h) of buffered solutions (pH 7.0) of each deoxynucleoside: dA, dT, dC, dG (5 x lo-‘M) in the presence of TB (lo-‘M), a decrease (30%) of dG absorption was detected (Table 1). Nevertheless, there were no changes in the absorption spectra of the other deoxynucleosides. Such results are consistent with a specific degradation of the dG. NaN, was used for the determination of the role of ‘02 in TB-sensitized dG photo-oxidation. The addition of NaN3 (lo-* M) to TB (lo-’ M)dG (5 x 10e5 M) solution results in a complete inhibition of dG photodegradation (see Table 1). Similar experiments with buffered solutions of dG alone, did not produce any absorption change. The irradiations were carried out at room temperature in a cylindrical glass cell of 2 cm pathlength and 5 cm3 volume. The light source was a 50 W (Osram HBO) high pressure Hg lamp. The

0

I 30 (min)

I 45

I

60

Fig. 3. Inhibitory effect of increasing concentrations of NaN, on the TRP (5 x 10m5M) photodegradation by TB (10e4 M) as a function of time of irradiation: (0) NaNS, 5 X 1O-4 M; (0) NaN,, lo-’ M; (W) NaN3, 5 x lo-’ M; (0) NaN3, 10m2M. The TRP photodegradation (0) in the

absence of inhibitor is also shown.

Technical Note

1267

Table 1. Maximum absorption wavelength (column 2) and inhibition percentages of photodegradation (column 3) for several deoxynucleoside solutions irradiated with red light in the presence of TB ( 10m5M) and NaNll (last row)

Irradiated sample TB+dA TB+dC TB+dT TB+dG TB + dG + NaN3

Deoxynucleoside absorption maximum (nm)

Relative rate of deoxynucleoside absorption decrease (%)

259.5 271.0 267.0 252.5 252.5

0 0 0 30 0

light was filtered by a glass filter to remove i.r. radiation and a red filter (600 nm
by grant PB87-0129 from Comisidn Investigadora

Departamento de Biologi’a (C-XV), Facultad de Ciencias, Universidad Authoma de Madrid, 28049-Madrid, Spain

de Ciencia y Tecnologfa

MAGDALENACAAETE* ANGELESVILLANUEVA

REFERENCES [l] J. Moan, Phorochem. Photobiol. 39, 445 (1984). [2] A. Seret, E. Gandin and A. Van De Vorst, Photobiochem. Phorobiophys. 12,259 (1986). [3] J. Cadet, M. Berger, C. Decarroz, J. R. Wagner, J. E. Van Lier, Y. M. Ginot and P. Vigny, Biochimie 68, 813 (1986). [4] J. M. Kelly, W. J. M. Van Der Putten and D. J. McConnell, Photochem. Phofobiol. 45, 167 (1987). [5] B. Gutter, W. T. Speck and H. S. Rosenkranz, Biochim. Eiophys. Acta 475, 307 (1977). [6] J. Cadet, C. Decarroz, S. Y. Wang and W. R. Widden, hr. J. Chem. 23,420 (1983).

* Author to whom correspondence

should be addressed.